A reactive selenium donor compound required both for the specific insertion of selenocysteine into proteins and for synthesis of selenouridine in tRNAs has been identified as selenophosphate using 31P NMR and radioisotope methodology. The overall reaction, ATP + selenide = selenophosphate + orthophosphate + AMP is catalyzed by selenophosphate synthetase. The reaction mechanism involves the intermediate formation of Enz-PP. Subsequent reaction with selenide forms selenophosphate containing the gamma phosphoryl group of ATP. The gene encoding selenoprotein A of clostridial glycine reductase was cloned, sequenced, and expressed in Escherichia coli. The isolated protein exhibited low activity in the glycine reductase assay. Moreover, full length immunologically reactive protein also was synthesized in the absence of selenium. Suppression and readthrough of the UGA codon by cysteyl-tRNA allowing insertion of cysteine (or occasionally selenocysteine when Se was supplied) was indicated. Random incorporation of either cysteine or selenocysteine at the UGA codon as an explanation of the low activity of the protein product was confirmed using a transformed E. coli mutant lacking selenophosphate synthetase. Protein containing only cysteine was inactive as a glycine reductase component. It appears that the E. coli translation system could not recognize the Clostridial nucleotide sequence required for specific insertion of selenocysteine at the UGA codon. The Se-carboxymethyl derivative of selenoprotein A, an intermediate in the glycine reductase reaction, is reductively cleaved by the protein C component giving an acetylthiol ester derivative of protein C. Enzyme inactivated by alkylation with [14C]iodoacetate was labeled in the 48 kDa subunit but not detectably in the 59 kDa subunit. Protein C is remarkably stable and active enzyme was recovered after heating in SDS. Subunits separated by SDS PAGE, when eluted from gel slices, renatured and recombined, exhibited detectable enzyme activity. Antibody production and detailed compositional studies of the subunits are in progress.